Method for automated, large-scale measurement of the molecular flux rates of the proteome or the organeome using mass spectrometry

ABSTRACT

Disclosed here is a method for measuring the kinetics (i.e., the molecular flux rates—synthesis and breakdown or removal rates) of a plurality of proteins or organic metabolites in living systems. The methods may be accomplished in a high-throughput, large-scale automated manner, by using existing mass spectrometric profiling techniques and art well known in the fields of static proteomics and static organeomics, without the need for additional biochemical preparative steps or analytic/instrumental devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/534,807 (now U.S. Pat. No. 8,129,335), filed Aug. 3, 2009, which is acontinuation of U.S. patent application Ser. No. 10/523,250, filed Jan.26, 2005, which is a U.S. National Phase Patent Application ofInternational Application No. PCT/US2003/023340, filed Jul. 25, 2003,which claims priority to U.S. Provisional Patent Application Ser. No.60/399,950 filed Jul. 30, 2002, all of which are hereby incorporated byreference in the present disclosure in their entireties.

FIELD OF THE INVENTION

The invention relates to methods for measuring molecular flux rates(synthesis and breakdown or input and removal rates from pools ofmolecules) in the proteome and the organeome (dynamic proteomics anddynamic organeomics, respectively) using mass spectrometry. The methodsdisclosed are capable of high-throughput, large-scale, automatedapplications. The methods are applicable to studies in genetics,functional genomics, drug discovery and development, drug toxicity,clinical diagnostics and patient management.

BACKGROUND OF THE INVENTION

Recent advances in the Human Genome project have, paradoxically, led tothe wide-spread recognition of the inadequacy of gene sequenceinformation by itself. Sequence information (i.e. structural genomics)is unlikely to generate insight into disease or normal physiologywithout better information concerning the functional consequence ofgenes. Higher levels of biological organization relative to genesequences include expressed mRNA levels, the expressed proteincomplement and concentrations of organic molecules in metabolicpathways. These levels of cellular organization have been called geneexpression profiling (transcriptomics), proteomics and organeomics,respectively. In aggregate, these can be seen as including thestructural biochemical phenotype (i.e. the complete complement ofmolecules present) in a cell or organism.

Gene expression profiling of mRNA has been achieved through thedevelopment of gene expression chips. Such chips are available fromcompanies such as Affymetrix. Enumerating the expressed genome (i.e. thecomplement of mRNA species), even in its entirety, however, does notultimately provide information about biochemical function (phenotype) ina living system. Although impressive as a technology, gene expressionchips do not solve the central problems of phenotype and function inbiochemistry, which relate to the flow of molecules through the complexinteractive network of proteins that comprise fully assembled livingsystems.

Other methods have focused on characterizing the complement of proteinsin a living system, i.e. the “proteome.” The most powerful technologyfor automated, large-scale characterization of expressed proteins(proteomics) to date has proven to be mass spectrometry. Massspectrometers have greatly simplified large-scale automated proteomeanalysis. Analogous mass spectrometric methods have been advanced forthe automated, large-scale characterization of organic metabolites(organeomics).

Many scientists in the pharmaceutical industry, including those ingenomics companies, are predicting a log-jam of potential drug leads andtargets that are under development. This log-jam arises frombottle-necks in the testing of phenotypic consequences of inhibitingparticular targets—i.e. the “tail-end” of drug development. The tail-endof drug development (target validation) has not received a similar pushfrom breakthrough technologies as the “front-end” (target identificationand identification of chemical modulators of targets) of drug discovery.

One of the central problems in this area relates to the absence ofroutine, high-throughput dynamic measurements in biology and medicine.Just as biochemical phenotype is recognized to be reducible to the flowof molecules through metabolic pathways in complex catalytic networks,it is also widely recognized that most diseases reduce to an alteredrate of a normal process. For example, atherogenesis reflects vascularwall proliferation and uptake of lipids; carcinogenesis reflects cellproliferation; infection can be characterized as microbial division,growth and death—this formulation is more informative than describingthese disorders as alterations of static measures (e.g. concentrationsof cholesterol, carcinogens, or bacteria). Yet rarely, if ever, arerates of biochemical processes measured in medical diagnostics. Staticmarkers of dynamic processes are often helpful and may be better thannothing, but they are not the true measure of disease activity ordisease risk. Nor do static measures allow for personalized biochemicalmonitoring. For example, each individual may have a differentrelationship between CD4 count and true turnover of T lymphocytes in HIVinfection, or between DNA-adducts and the true risk of cancer, orbetween LDL cholesterol and the true rate of atherogenesis. In the finalanalysis, kinetic questions must be addressed by direct kineticmeasurements.

Thus, the current art in mass spectrometric proteomics and organeomicsis characterized by a shared and fundamental limitation: the informationis static, not dynamic. Missing from both static proteomics and staticorganeomics is kinetics: fluxes into and out of the pools of moleculesthat are present in the system. Kinetics or dynamics differ from staticsin the fundamental respect that the dimension of time is induded.Kinetics refers to the study of time-related changes in moleculeswhereas the concentrations of proteins or organic molecules determinedin static measurements do not provide any information about their ratesof change over time. Although the current techniques of static proteomeand organeome characterization can provide a snapshot of what ispresent, these techniques cannot provide information concerning flows ofmolecules through the system (kinetics).

Thus, there is a tremendous need for the large scale determination ofmolecular flux rates of a plurality of proteins or organicmetabolites—i.e. “dynamic proteomics” and “dynamic organeomics”.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed to amethod of determining the molecular flux rates (i.e., the rates ofsynthesis or breakdown of a plurality of proteins in all or a portion ofthe proteome of a cell, tissue or organism). One or more isotope-labeledprotein precursors are administered to a cell, tissue or organism for aperiod of time sufficient for one or more isotope labels to beincorporated into a plurality of proteins in the proteome or portionthereof of the cell, tissue or organism. The proteome or portion thereofare then obtained from the cell, tissue, or organism. A plurality ofmass isotopomeric envelopes representing individual proteins in theproteome or portion of the proteome are then identified by massspectrometry. In addition, the relative and absolute mass isotopomerabundances of the ions within the isotopomeric envelope corresponding toeach identified protein are quantified by mass spectrometry. Theserelative and absolute mass isotopomer abundances allow the molecularflux rates of each identified protein to be calculated and the molecularflux rates of the plurality of proteins thereby to be determined.

In one aspect, the administering step may be continuous. The proteinprecursors may also be administered at regular measured intervals. Theprotein precursors may also be administered orally. The method mayinclude the additional step of discontinuing the administering step.

The one or more protein precursors may be an amino acid, or may includeone or more precursors such as H₂O, CO₂, NH₃, and HCO₃. Isotope labelmay include one or more isotopes such as ²H, ¹³C, ¹⁵N, ¹⁸O, ³³S and ³⁴S.In a particular embodiment, the isotope label may be ²H.

In another aspect, the proteins may be modified prior to the measuringstep. The modification may be by any method known in the art, such asbiochemically degrading the proteins, or by chemically altering theproteins.

In a further aspect, the individual proteins may also be identified byboth chromatography and mass spectrometry. In an additional embodiment,the plurality of proteins may also include the entire proteome of thecell, tissue, or organism. The calculated molecular flux rates of theproteins may be displayed after calculation.

The organism may be any organism in the art from cells in culture toliving animals. In one aspect, the organism is a human.

In another aspect, the methods may further include administering adiagnostic or therapeutic agent to the cell, tissue, or organism priorto administering the isotope labeled precursor. The invention is alsodirected to a method of determining the effect of a diagnostic ortherapeutic agent on a cell, tissue, or organism, by determining themolecular flux rates of a plurality of proteins in the cell, tissue, ororganisms, administering the agent and again determining the molecularflux rates of the plurality of proteins in the cell, tissue or organism.The discovery and development of drugs can be achieved or facilitated bythis means.

The methods may also include determining the effects of one or moregenes on the molecular flux rates of synthesis of a plurality ofproteins in a cell, tissue, or organism by determining the molecularflux rates of a plurality of proteins in a first population of one ormore cells, tissues, or organisms that has one or more genes,determining the molecular flux rates on the plurality of proteins in asecond population of one or more cells, tissues, or organisms that doesnot have the one or more genes, and comparing the molecular flux ratesin the first and second populations to determine the effect of one ormore genes on the molecular flux rates of a plurality of proteins.

In another aspect, the invention is drawn to determining the molecularflux rates of a plurality of organic metabolites in all or a portion ofthe organeome of a cell, tissue or organism. One or more isotope-labeledorganic metabolites or organic metabolite precursors are administered tothe cell, tissue or organism for a period of time sufficient for one ormore isotope labels to be incorporated into a plurality of organicmetabolites in the organeome or portion thereof of the cell, tissue ororganism. The organeome or portion thereof is obtained from the cell,tissue, or organism. A plurality of mass isotopomeric envelopes of ionsrepresenting individual organic metabolites in the organeome or portionthereof are identified by mass spectrometry. In addition, the relativeand absolute mass isotopomer abundances of the ions within the isotopicenvelopes corresponding to each identified organic metabolite arequantified by mass spectrometry. These relative and absolute massisotopomer abundances allow the rates of synthesis or removal of eachidentified organic metabolite to be calculated, and the molecular fluxrates of the plurality of organic metabolites thereby to be determined.

In one aspect, one or more organic metabolites or organic metaboliteprecursors include one or more of H₂O, CO₂, NH₃, HCO₃, amino acids,monosaccharides, carbohydrates, lipids, fatty acids, nucleic acids,glycolytic intermediates, acetic acid, and tricarboxylic acid cycleintermediates. In another aspect, the isotope label includes ²H, ¹³C,¹⁵N, ¹⁸O, ³³S or ³⁴S. The plurality of organic metabolite precursors mayinclude the entire organeome. The organism may be any known organism,including a human. In a particular embodiment, the isotope label may be²H.

In another aspect, the administration of precursors may be continuous.Alternatively, the precursor may be administered at regular measuredintervals. The one or more organic metabolites or organic metaboliteprecursors may be administered orally. Further, the method may includethe additional step of discontinuing administration of the labeledprecursor.

In an additional aspect, the method may include modifying organicmetabolites prior to introduction into the mass spectrometer. Themodification may be any method known in the art, such as biochemicallydegrading the organic metabolites or chemically altering the organicmetabolites.

Individual organic metabolites in the organeome or portion thereof maybe identified by mass spectrometry, and/or by chromatography. Thecalculated synthesis or removal rates of the plurality of organicmetabolites may be displayed.

In another aspect, the invention is drawn to methods of administering adiagnostic or therapeutic agent to the cell, tissue, or organism priorto administering the precursor. In one embodiment, the invention isdrawn to a method of determining the effect of a diagnostic ortherapeutic agent on a cell, tissue, or organism by determining therates of synthesis or removal of a plurality of organic metabolites inthe cell, tissue, or organism, administering an agent, and determiningthe rates of synthesis or removal on the plurality of organicmetabolites in the cell, tissue or organism. By this means, drugdiscovery and development may be facilitated or achieved.

In another aspect, the invention is drawn to a method of determining theeffects of one or more genes on the molecular flux rates of a pluralityof organic metabolites in a cell, tissue, or organism by determining themolecular flux rates of a plurality of organic metabolites in a firstpopulation of one or more cells, tissues, or organisms having One ormore genes; determining the molecular flux rates of the plurality oforganic metabolites in a second population of one or more cells,tissues, or organisms that do not include the one or more genes, andcomparing the molecular flux rates of said plurality of organicmetabolites in the first and second populations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram illustrating the organization ofcomplex biological systems.

FIG. 2 shows levels of functional genomics.

FIG. 3 shows techniques for measuring dynamic proteomics.

FIG. 4 depicts pathways of labeled hydrogen exchange from labeled waterinto selected free amino acids. Two nonessential amino adds (alanine,glycine) and an essential amino acid (leucine) are shown, by way ofexample. Alanine and glycine are presented in FIG. 4A. Leucine ispresented in FIG. 4B. Abbreviations: TA, transaminase; PEP-CK,phosphoenol-pyruvate carboxykinase; TCAC, tricarboxylic acid cycle;STHM, serine tetrahydrofolate methyl transferase. FIG. 4C depicts H₂ ¹⁸Olabeling of free amino acids for protein synthesis.

DETAILED DESCRIPTION OF THE INVENTION

The inventor has discovered a method of determining molecular flux rates(i.e., synthesis, breakdown and removal rates) of a plurality ofproteins or a plurality of organic metabolites. First, anisotope-labeled precursor molecule is administered to a cell, tissue, ororganism. The molecular flux rates of a plurality of proteins or aplurality of organic metabolites are then determined.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) 3. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Cabs, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); and Mass isotopomer distribution analysis ateight years: theoretical, analytic and experimental considerations byHellerstein and Neese (Am J Physiol 276 (Endocrinol Metab. 39)E1146-E1162, 1999). Furthermore, procedures employing commerciallyavailable assay kits and reagents will typically be used according tomanufacturer-defined protocols unless otherwise noted.

II. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, Massisotopomer distribution analysis at eight years: theoretical, analyticand experimental considerations by Hellerstein and Neese (Am J Physiol276 (Endocrinol Metab. 39) E1146-E1162, 1999). As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

“Molecular flux rates” refers to the rate of synthesis and/or breakdownof a protein and/or organic metabolite. “Molecular flux rates” alsorefers to a protein and/or organic metabolite's input into or removalfrom a pool of molecules, and is therefore synonymous with the flow intoand out of said pool of molecules.

“Isotopologues” refer to isotopic homologues or molecular species thathave identical elemental and chemical compositions but differ inisotopic content (e.g., CH₃NH₂ vs. CH₃NHD in the example above).Isotopologues are defined by their isotopic composition, therefore eachisotopologue has a unique exact mass but may not have a uniquestructure. An isotopologue usually includes of a family of isotopicisomers (isotopomers) which differ by the location of the isotopes onthe molecule (e.g., CH₃NHD and CH₂DNH₂ are the same isotopologue but aredifferent isotopomers).

“Isotope-labeled water” includes water labeled with one or more specificheavy isotopes of either hydrogen or oxygen. Specific examples ofisotope-labeled water include ²H₂O, ³H₂O, and H₂ ¹⁸O.

“Protein Precursor” refers to any organic or inorganic molecule orcomponent thereof, wherein one or more atoms of which are capable ofbeing incorporated into protein molecules in cell, tissue, organism, orother biological system, through the biochemical processes of the cell,tissue, or organism. Examples of protein precursors include, but are notlimited to, amino acids, H₂O, CO₂, NH₃, and HCO₃.

“Isotope Labeled protein precursor” refers to a protein precursor thatcontains an isotope of an element that differs from the most abundantisotope of the element present in nature, cells, tissue, or organisms.The isotope label may include specific heavy isotopes of elementspresent in biomolecules, such as ²H, ¹³C, ¹⁵N, ¹⁸O, ³³S, ³⁴S, or maycontain other isotopes of elements present in biomolecules such as ³H,¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I. Isotope labeled protein precursors include; butare not limited to ²H₂0, ¹⁵NH₃, ¹³CO₂, H¹³C0₃, ²H-labeled amino acids,¹³C labeled amino acids, ¹⁵N labeled amino acids, ¹⁸O labeled aminoacids, ³⁴S or ³³S labeled amino acids, ³H₂O ³H-labeled amino acids, and¹⁴C labeled amino acids.

“Isotope-labeled organic metabolite precursors” refer to an organicmetabolite precursor that contains an isotope of an element that differsfrom the most abundant isotope of said element present in nature orcells, tissues, or organisms. Isotopic labels include specific heavyisotopes of elements, present in biomolecules, such as ²H, ¹³C, ¹⁵N,¹⁸O, ³⁵S, ³⁴S, or may contain other isotopes of elements present inbiomolecules, such as ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I. Isotope labeled organicmetabolite precursors include but are not limited to ²H₂O, ¹⁵NH₃, ¹³CO₂,H¹³CO₃, ²H-labeled amino acids, ¹³C-labeled amino acids, ¹⁵N-labeledamino acids, ¹⁸O-labeled amino acids, ³³S or ³⁴S-labeled amino acids,³H₂O, ³H-labeled amino acids, ¹⁴C-labeled amino acids, ¹⁴CO₂, andH¹⁴CO₂.

“Partially purifying” refers to methods of removing one or morecomponents of a mixture of other similar compounds. For example,“partially purifying a protein” refers to removing one or more proteinsfrom a mixture of one or more proteins.

“Isolating” refers to separating one compound from a mixture ofcompounds. For example, “isolating a protein” refers to separating onespecific protein from all other proteins in a mixture of one or moreproteins.

A “biological sample” encompasses any sample obtained from a cell,tissue, or organism. The definition encompasses blood and other liquidsamples of biological origin, that are accessible from an organismthrough sampling by minimally invasive or non-invasive approaches (e.g.,urine collection, blood drawing, needle aspiration, and other proceduresinvolving minimal risk, discomfort or effort). The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or organicmetabolites. The term “biological sample” also encompasses a clinicalsample such as serum, plasma, other biological fluid, or tissue samples,and also includes cells in culture, cell supematants and cell lysates.

“Biological fluid” refers, but is not limited to, urine, blood,interstitial fluid, edema fluid, saliva, lacrimal fluid, inflammatoryexudates, synovial fluid, abscess, empyema or other infected fluid,cerebrospinal fluid, sweat, pulmonary secretions (sputum), seminalfluid, feces, bile, intestinal secretions, or other biological fluid.

“Exact mass” refers to mass calculated by summing the exact masses ofall the isotopes in the formula of a molecule (e.g. 32.04847 forCH₃NHD).

“Nominal mass” refers to the integer mass obtained by rounding the exactmass of a molecule.

“Mass isotopomer” refers to family of isotopic isomers that is groupedon the basis of nominal mass rather than isotopic composition. A massisotopomer may comprise molecules of different isotopic compositions,unlike an isotopologue (e.g. CH₃NHD, ¹³CH₃NH₂, CH₃ ¹⁵NH₂ are part of thesame mass isotopomer but are different isotopologues). In operationalterms, a mass isotopomer is a family of isotopologues that are notresolved by a mass spectrometer. For quadrupole mass spectrometers, thistypically means that mass isotopomers are families of isotopologues thatshare a nominal mass. Thus, the isotopologues CH₃NH₂ and CH₃NHD differin nominal mass and are distinguished as being different massisotopomers, but the isotopologues CH₃NHD, CH₂DNH₂, ¹³CH₃NH₂, and CH₃¹⁵NH₂ are all of the same nominal mass and hence are the same massisotopomers. Each mass isotopomer is therefore typically composed ofmore than one isotopologue and has more than one exact mass. Thedistinction between isotopologues and mass isotopomers is useful inpractice because all individual isotopologues are not resolved usingquadrupole mass spectrometers and may not be resolved even using massspectrometers that produce higher mass resolution, so that calculationsfrom mass spectrometric data must be performed on the abundances of massisotopomers rather than isotopologues. The mass isotopomer lowest inmass is represented as M₀; for most organic molecules, this is thespecies containing all ¹²C, ¹H, ¹⁶O, ¹⁴N, etc. Other mass isotopomersare distinguished by their mass differences from M₀ (M₁, M₂, etc.). Fora given mass isotopomer, the location or position of isotopes within themolecule is not specified and may vary (i.e. “positional isotopomers”are not distinguished).

“Mass isotopomer envelope” refers to the set of mass isotopomersassociated with a molecule or ion fragment.

“Mass isotopomer pattern” refers to a histogram of the abundances of themass isotopomers of a molecule. Traditionally, the pattern is presentedas percent relative abundances where all of the abundances arenormalized to that of the most abundant mass isotopomer; the mostabundant isotopomer is said to be 100%. The preferred form forapplications involving probability analysis, such as mass isotopomerdistribution analysis (MIDA), however, is proportion or fractionalabundance, where the fraction that each species contributes to the totalabundance is used. The term “isotope pattern” may be used synonomouslywith the term “mass isotopomer pattern.”

“Monoisotopic mass” refers to the exact mass of the molecular speciesthat contains all ¹H, ¹²C, ¹⁴N, ¹⁶O, ³²S, etc. For isotopologuescomposed of C, H, N, O, P, S, F, CI, Br, and I, the isotopic compositionof the isotopologue with the lowest mass is unique and unambiguousbecause the most abundant isotopes of these elements are also the lowestin mass. The monoisotopic mass is abbreviated as m₀ and the masses ofother mass isotopomers are identified by their mass differences from m₀(m₁, m₂, etc.).

“Isotopically perturbed” refers to the state of an element or moleculethat results from the explicit incorporation of an element or moleculewith a distribution of isotopes that differs from the distribution foundin nature, whether a naturally less abundant isotope is present inexcess (enriched) or in deficit (depleted).

“Monomer” refers to a chemical unit that combines during the synthesisof a polymer and which is present two or more times in the polymer.

“Polymer” refers to a molecule synthesized from and containing two ormore repeats of a monomer.

“Protein” refers to a polymer of amino acids. As used herein, a“protein” may be to a long amino acid polymers as well as short polymerssuch as peptides.

“Proteome” refers to the complement of proteins expressed by a cell,tissue or organism under a specific set of conditions.

“Static proteomics” refers to current mass spectrometric techniqueswell-known in the art for characterizing the protein complementexpressed in a cell, tissue or organism by their concentrations orlevels but not as the rates of synthesis or breakdown (fluxes) of theseproteins (to be contrasted with Dynamic Proteomics).

“Dynamic proteomics” refers to mass spectrometric techniques forcharacterizing the rates of synthesis and/or breakdown (fluxes) of theproteins in a proteome.

“Organic metabolite” refers to any organic molecule involved inmetabolism in a cell, tissue, or organism. Organic metabolites mayinclude, but are not limited to, amino acids, sugars, sugar alcohols,organic acids, sterols, and nucleotide bases.

“Organeome” refers to the population of organic molecules present in acell, tissue or organism. “Organeome” may also refer to the populationof organic molecules present in a cell, tissue or organism under aspecific set of conditions.

“Static organeomics” refers to current mass spectrometric techniqueswell known in the art for characterizing the complement of organicmolecules or metabolites present in a cell, tissue or organism by theirconcentrations or levels, but not by the rates of synthesis or breakdownof these organic molecules or metabolites.

“Dynamic organeomics” refers mass spectrometric techniques forcharacterizing the rates of synthesis and/or breakdown of the organicmolecules or metabolites in an organeome.

“Organic metabolite precursor” refers to an organic or inorganicmolecule capable of entering into cellular pools of organic metaboliteseither directly or by prior transformation.

III. Methods of the Invention

The present invention is directed to methods of determining themolecular flux rates of a plurality of proteins in all or a portion ofthe proteome of a cell, tissue or organism. First, one or moreisotope-labeled protein precursors are administered to a cell, tissue ororganism for a period of time sufficient to be incorporated into aplurality of proteins in the proteome or portion thereof. The proteomeor portion thereof is obtained from the cell, tissue, or organism, and aplurality of individual proteins are identified by mass spectrometry.The relative and absolute mass isotopomer abundances of the ions withinthe isotopomeric envelope corresponding to each identified protein orpeptide are quantified by mass spectrometry, and the molecular fluxrates of each identified protein or peptide of said plurality ofproteins can be determined.

The same methodology may be applied to determine the molecular fluxrates of a plurality of organic metabolites in all or a portion of theorganeome.

The organization of complex biological systems is illustrated in FIG. 1.The levels of functional genomics are illustrated in FIG. 2. The presentinvention is directed to methods of measuring, analyzing, quantitating,qualitating and interpreting dynamic proteomic measurements and dynamicorganeomic measurements. (See FIG. 3).

Administering Isotope-Labeled Precursor(s)

As a first step in the methods of the invention, isotope-labeledprecursors are administered.

A. Administering an Isotope-Labeled Precursor Molecule

1. Labeled Precursor Molecules

a. Isotope Labels

The first step in measuring molecular flux rates involves administeringan isotope-labeled precursor molecule to a cell, tissue, or organism.The isotope labeled precursor molecule may be a stable isotope orradioisotope. Isotope labels that can be used include, but are notlimited to, ²H, ¹³C, ¹⁵N, ¹⁸O, ³H, ¹⁴C, ³⁵S, ³²P, ¹²⁵I, ¹³¹I, or otherisotopes of elements present in organic systems.

In one embodiment, the isotope label is ²H.

b. Precursor Molecules

The precursor molecule may be any molecule having an isotope label thatis incorporated into a protein or organic metabolite. Isotope labels maybe used to modify all precursor molecules disclosed herein to formisotope-labeled precursor molecules.

The entire precursor molecule may be incorporated into one or moreproteins and/or organic metabolites. Alternatively, a portion of theprecursor molecule may be incorporated into one or more proteins and/ororganic metabolite.

Precursor molecules may include, but not limited to, CO₂, NH₃, glucose,lactate, H₂O, acetate, and fatty acids.

i. Protein Precursors

A protein precursor molecule may be any protein precursor molecule knownin the art. These precursor molecules may be CO₂, NH₃, glucose, lactate,H₂O, acetate, and fatty acids.

Precursor molecules of proteins may also include one or more aminoacids. The precursor may be any amino acid. The precursor molecule maybe a singly or multiply deuterated amino acid. For example, theprecursor molecule may be one or more of ¹³C-lysine, ¹⁵N-histidine,¹³C-serine, ¹³C-glycine, ²H-leucine, ¹⁵N-glycine, ¹³C-leucine,²H₅-histidine, and any deuterated amino acid. Labeled amino acids may beadministered, for example, undiluted or diluted with non-labeled aminoacids. All isotope labeled precursors may be purchased commercially, forexample, from Cambridge Isotope Labs (Andover, Mass.).

Protein precursor molecules may also include any precursor forpost-translational or pre-translationally modified amino acids. Theseprecursors include but are not limited to precursors of methylation suchas glycine, serine or H₂O; precursors of hydroxylation, such as H₂O orO₂; precursors of phosphorylation, such as phosphate, H₂O or O₂;precursors of prenylation, such as fatty acids, acetate, H₂O, ethanol,ketone bodies, glucose, or fructose; precursors of carboxylation, suchas CO₂, O₂, H₂O, or glucose; precursors of acetylation, such as acetate,ethanol, glucose, fructose, lactate, alanine, H₂O, CO₂, or O₂; and otherpost-translational modifications known in the art.

The degree of labeling present in free amino acids may be determinedexperimentally, or may be assumed based on the number of labeling sitesin an amino acid. For example, when using hydrogen isotopes as a label,the labeling present in C—H bonds of free amino acid or, morespecifically, in tRNA-amino acids, during exposure to ²H₂O in body watermay be identified. The total number of C—H bonds in each non essentialamino acid is known—e.g. 4 in alanine, 2 in glycine, etc.

The precursor molecule for proteins may be water. The hydrogen atoms onC—H bonds are the hydrogen atoms on amino acids that are useful formeasuring protein synthesis from ²H₂O since the O—H and N—H bonds ofproteins are labile in aqueous solution. As such, the exchange of²H-label from ²H₂O into O—H or N—H bonds occurs without the synthesis ofproteins from free amino acids as described above. C—H bonds undergoincorporation from H₂O into free amino acids during specificenzyme-catalyzed intermediary metabolic reactions (FIG. 4). The presenceof ²H-label in C—H bonds of protein-bound amino acids after ²H₂Oadministration therefore means that the protein was assembled from aminoacids that were in the free form during the period of ²H₂O exposure—i.e.that the protein is newly synthesized. Analytically, the amino addderivative used must contain all the C—H bonds but must remove allpotentially contaminating N—H and O—H bonds.

Hydrogen atoms from body water may be incorporated into free aminoacids. ²H or ³H from labeled water can enter into free amino adds in thecell through the reactions of intermediary metabolism, but ²H or ³Hcannot enter into amino acids that are present in peptide bonds or thatare bound to transfer RNA. Free essential amino acids may incorporate asingle hydrogen atom from body water into the α-carbon C—H bond, throughrapidly reversible transamination reactions (FIG. 4). Free non-essentialamino adds contain a larger number of metabolically exchangeable C—Hbonds, of course, and are therefore expected to exhibit higher isotopicenrichment values per molecule from ²H₂O in newly synthesized proteins(FIGS. 4A-B).

One of skill in the art will recognize that labeled hydrogen atoms frombody water may be incorporated into other amino acids via otherbiochemical pathways. For example, it is known in the art that hydrogenatoms from water may be incorporated into glutamate via synthesis of theprecursor α-ketoglutrate in the citric acid cycle. Glutamate, in turn,is known to be the biochemical precursor for glutamine, proline, andarginine. By way of another example, hydrogen atoms from body water maybe incorporated into post-translationally modified amino acids, such asthe methyl group in 3-methyl-histine, the hydroxyl group inhydroxyproline or hydroxylysine, and others. Other amino adds synthesispathways are known to those of skill in the art.

Oxygen atoms (H₂ ¹⁸O) may also be incorporated into amino acids throughenzyme-catalyzed reactions. For example, oxygen exchange into thecarboxylic acid moiety of amino acids may occur during enzyme catalyzedreactions. Incorporation of labeled oxygen into amino acids is known toone of skill in the art as illustrated in FIG. 4C. Oxygen atoms may alsobe incorporated into amino acids from ¹⁸O₂ through enzyme catalyzedreactions (including hydroxyproline, hydroxylysine or otherpost-translationally modified amino acids).

Hydrogen and oxygen labels from labeled water may also be incorporatedinto amino acids through post-translational modifications. In oneembodiment, the post-translational modification may already includelabeled hydrogen or oxygen through biosynthetic pathways prior topost-translational modification. In another embodiment, thepost-translational modification may incorporate labeled hydrogen,oxygen, carbon, or nitrogen from metabolic derivatives involved in thefree exchange labeled hydrogens from body water, either before or afterpost-translational modification step (e.g. methylation, hydroxylation,phosphorylation, prenylation, sulfation, carboxylation, acetylation orother known post-translational modifications).

Protein precursors for that are suitable for administration into asubject include, but are not limited to H₂O, CO₂, NH₃ and HCO₃, inaddition to the standard amino acids found in proteins.

ii. Precursors of Organic Metabolites

Precursors of organic metabolites may be any precursor molecule capableof entering into the organic metabolite pathway. Organic metabolites andorganic metabolite precursors include H₂O, CO₂, NH₃, HCO₃, amino acids,monosaccharides, carbohydrates, lipids, fatty acids, nucleic acids,glycolytic intermediates, acetic acid, and tricarboxylic acid cycleintermediates.

Organic metabolite precursors may also be administered directly. Massisotopes may be useful in mass isotope labeling of protein or organicmetabolite precursors include, but are not limited to ²H, ¹³C, ¹⁵N, ¹⁸O,³³S and ³⁴S. It is often desirable, in order to avoid metabolic loss ofisotope labels, that the isotope-labeled atom(s) be relativelynon-labile or at least behave in a predictable manner within thesubject. By administering the isotope-labeled precursors to thebiosynthetic pool, the isotope-labeled precursors can become directlyincorporated into organic metabolites formed in the pool.

iii. Water as a Precursor Molecule

Water is a precursor of proteins and many organic metabolites. As such,labeled water may serve as a precursor in the methods taught herein.

Labeled water may be readily obtained commercially. For example, ²H₂Omay be purchased from Cambridge Isotope Labs (Andover, Mass.), and ³H₂Omay be purchased, e.g., from New England Nuclear, Inc. In general, ²H₂Ois non-radioactive and thus, presents fewer toxicity concerns thanradioactive ³H₂O. ²H₂O may be administered, for example, as a percent oftotal body water, e.g., 1% of total body water consumed (e.g., for 3litres water consumed per day, 30 microliters ²H₂O is consumed). If ³H₂Ois utilized, then a non-toxic amount, which is readily determined bythose of skill in the art, is administered.

Relatively high body water enrichments of ²H₂O (e.g., 1-10% of the totalbody water is labeled) may be achieved relatively inexpensively usingthe techniques of the invention. This water enrichment is relativelyconstant and stable as these levels are maintained for weeks or monthsin humans and in experimental animals without any evidence of toxicity.This finding in a large number of human subjects (>100 people) iscontrary to previous concerns about vestibular toxicities at high dosesof ²H₂O. The Applicant has discovered that as long as rapid changes inbody water enrichment are prevented (e.g., by initial administration insmall, divided doses), high body water enrichments of ²H₂O can bemaintained with no toxicities. For example, the low expense ofcommercially available ²H₂O allows long-term maintenance of enrichmentsin the 1-5% range at relatively low expense (e.g., calculations reveal alower cost for 2 months labeling at 2% ²H₂O enrichment, and thus 7-8%enrichment in the alanine precursor pool (FIG. 4A-B), than for 12 hourslabeling of ²H-leucine at 10% free leucine enrichment, and thus 7-8%enrichment in leucine precursor pool for that period).

Relatively high and relatively constant body water enrichments foradministration of H₂ ¹⁸O may also be accomplished, since the ¹⁸O isotopeis not toxic, and does not present a significant health risk as a result(FIG. 4C).

iv. Modes of Administering Precursors of Proteins and OrganicMetabolites

Modes of administering the one or more isotope-labeled precursors mayvary, depending upon the absorptive properties of the isotope-labeledprecursor and the specific biosynthetic pool into which each compound istargeted. Precursors may be administered to organisms, plants andanimals including humans directly for in vivo analysis. In addition,precursors may be administered in vitro to living cells. Specific typesof living cells include hepatocytes, adipocytes, myocytes, fibroblasts,neurons, pancreatic β-cells, intestinal epithelial cells, leukocytes,lymphocytes, erythrocytes, microbial cells and any other cell-type thatcan be maintained alive and functional in vitro.

Generally, an appropriate mode of administration is one that produces asteady state level of precursor within the biosynthetic pool and/or in areservoir supplying such a pool for at least a transient period of time.Intravenous or oral routes of administration are commonly used toadminister such precursors to organisms, including humans. Other routesof administration, such as subcutaneous or intra-muscularadministration, optionally when used in conjunction with slow releaseprecursor compositions, are also appropriate. Compositions for injectionare generally prepared in sterile pharmaceutical excipients.

B. Obtaining a Plurality of Proteins or Organic Metabolites

In practicing the method of the invention, in one aspect, proteins andorganic metabolites are obtained from a cell, tissue, or organismaccording to the methods known in the art. The methods may be specificto the proteins or organic metabolites of interest. Proteins and organicmetabolites of interest may be isolated from a biological sample.

A plurality of proteins or a plurality of organic metabolites may beacquired from the cell, tissue, or organism. The one or more biologicalsamples may be obtained, for example, by blood draw, urine collection,biopsy, or other methods known in the art. The one or more biologicalsample may be one or more biological fluids. The protein or organicmetabolite may also be obtained from specific organs or tissues, such asmuscle, liver, adrenal tissue, prostate tissue, endometrial tissue,blood, skin, and breast tissue. Proteins or organic metabolites may beobtained from a specific group of cells, such as tumor cells orfibroblast cells. Proteins or organic metabolites also may be obtained,and optionally partially purified or isolated, from the biologicalsample using standard biochemical methods known in the art.

The frequency of biological sampling can vary depending on differentfactors. Such factors include, but are not limited to, the nature of theproteins or organic metabolites, ease and safety of sampling, synthesisand breakdown/removal rates of the proteins or organic metabolites fromwhich it was derived, and the half-life of a therapeutic agent orbiological agent.

The proteins or organic metabolites may also be purified partially, oroptionally, isolated, by conventional purification methods includinghigh pressure liquid chromatography (HPLC), fast performance liquidchromatography (FPLC), chemical extraction, thin layer chromatography,gas chromatography, gel electrophoresis, and/or other separation methodsknown to those skilled in the art.

In another embodiment, the proteins or organic metabolites may behydrolyzed or otherwise degraded to form smaller molecules. Hydrolysismethods include any method known in the art, including, but not limitedto, chemical hydrolysis (such as acid hydrolysis) and biochemicalhydrolysis (such as peptidase degradation). Hydrolysis or degradationmay be conducted either before or after purification and/or isolation ofthe proteins or organic metabolites. The proteins or organic metabolitesalso may be partially purified, or optionally, isolated, by conventionalpurification methods including high performance liquid chromatography(HPLC), fast performance liquid chromatography (FPLC), gaschromatography, gel electrophoresis, and/or any other methods ofseparating chemical and/or biochemical compounds known to those skilledin the art.

C. Analysis

Presently available technologies (static proteomics) used to profiledifferences in expressed proteins measure only protein levels(concentrations) in a cell and do so at one point in time. Approachesinclude Global Proteome Mapping (using 2D technology and MassSpectrometry), Differential Expression Analysis (using 2D/DIGEtechnology and Mass Spectrometry), and Expression Analysis (2D/DIGE,Mass Spectrometry and LC-based, or other novel technology). While RNAand protein expression “chips” can be used to rapidly detectbiologically mediated resistance to a therapeutic agent in a variety ofdisease states, they fail to determine the rate of change of geneexpression or protein turnover. The methods of the present inventionallow determination of the rates of gene expression and molecular fluxrates of a plurality of proteins, as well as the molecular flux rates ofa plurality of organic metabolites, and their changes over time.

Mass Spectrometry

Isotopic enrichment in proteins and organic metabolites can bedetermined by various methods such as mass spectrometry, including butnot limited to gas chromatography-mass spectrometry (GC-MS),isotope-ratio mass spectrometry, GC-isotope ratio-combustion-MS,GC-isotope ratio-pyrrolysis-MS, liquid chromatography-MS, electrosprayionization-MS, matrix assisted laser desorption-time of flight-MS,Fourier-transform-ion-cyclotron-resonance-MS, and cycloidal-MS.

Mass spectrometers convert molecules such as proteins and organicmetabolites into rapidly moving gaseous ions and separate them on thebasis of their mass-to-charge ratios. The distributions of isotopes orisotopologues of ions, or ion fragments, may thus be used to measure theisotopic enrichment in a plurality of proteins or organic metabolites.

Generally, mass spectrometers include an ionization means and a massanalyzer. A number of different types of mass analyzers are known in theart. These include, but are not limited to, magnetic sector analyzers,electrospray ionization, quadrupoles, ion traps, time of flight massanalyzers, and Fourier transform analyzers.

Mass spectrometers may also include a number of different ionizationmethods. These include, but are not limited to, gas phase ionizationsources such as electron impact, chemical ionization, and fieldionization, as well as desorption sources, such as field desorption,fast atom bombardment, matrix assisted laser desorption/ionization, andsurface enhanced laser desorption/ionization.

In addition, two or more mass analyzers may be coupled (MS/MS) first toseparate precursor ions, then to separate and measure gas phase fragmentions. These instruments generate an initial series of ionic fragments ofa protein, and then generate secondary fragments of the initial ions.The resulting overlapping sequences allows complete sequencing of theprotein, by piecing together overlaying “pieces of the puzzle”, based ona single mass spectrometric analysis within a few minutes (plus computeranalysis time).

The MS/MS peptide fragmentation patterns and peptide exact molecularmass determinations generated by protein mass spectrometry provideunique information regarding the amino acid sequence of proteins andfind use in the present invention. An unknown protein can be sequencedand identified in minutes, by a single mass spectrometric analytic run.The library of peptide sequences and protein fragmentation patterns thatis now available provides the opportunity to identify components ofcomplex proteome mixtures with near certainty.

Different ionization methods are also known in the art. One key advancehas been the development of techniques for ionization of large,non-volatile macromolecules including proteins and polynucleotides.Techniques of this type have included electrospray ionization (ESI) andmatrix assisted laser desorption (MALDI). These have allowed MS to beapplied in combination with powerful sample separation introductiontechniques, such as liquid chromatography and capillary zoneelectrophoresis.

In addition, mass spectrometers may be coupled to separation means suchas gas chromatography (GC) and high performance liquid chromatography(HPLC). In gas-chromatography mass-spectrometry (GC/MS), capillarycolumns from a gas chromatograph are coupled directly to the massspectrometer, optionally using a jet separator. In such an application,the gas chromatography (GC) column separates sample components from thesample gas mixture and the separated components are ionized andchemically analyzed in the mass spectrometer.

When GC/MS (or other mass spectrometric modalities that analyze ions ofproteins and organic metabolites, rather than small inorganic gases) isused to measure mass isotopomer abundances of organic molecules,hydrogen-labeled isotope incorporation from isotope-labeled water isamplified 3 to 7-fold, depending on the number of hydrogen atomsincorporated into the organic molecule from isotope-labeled water invivo.

In general, in order to determine a baseline mass isotopomer frequencydistribution for the protein, such a sample is taken before infusion ofan isotopically labeled precursor. Such a measurement is one means ofestablishing in the cell, tissue or organism, the naturally occurringfrequency of mass isotopomers of the protein. When a cell, tissue ororganism is part of a population of subjects having similarenvironmental histories, a population isotopomer frequency distributionmay be used for such a background measurement. Additionally, such abaseline isotopomer frequency distribution may be estimated, using knownaverage natural abundances of isotopes. For example, in nature, thenatural abundance of ¹³C present in organic carbon in 1.11%. Methods ofdetermining such isotopomer frequency distributions are discussed below.Typically, samples of the protein are taken prior to and followingadministration of an isotopically labeled precursor to the subject andanalyzed for isotopomer frequency as described below. Similarconsiderations apply to the isolation of organic molecules for DynamicOrganeomics.

Thus, a single analysis of even an enormously complex mixture ofproteins (that has been subjected to proteolytic cleavage or analyzeddirectly) can uniquely identify peptides representing thousands ofexpressed proteins.

Proteins may also be detected using protein chips. Several commercial“protein chip” equivalents are now marketed, using mass spectrometry(e.g. Ciphergen Biosystems). The efficiency of peptide sequencedetermination by mass analysis, combined with powerful ion fragmentationtechnology (MS/MS instruments) and/or peptide generating biochemicalmethods (e.g. proteolysis), improvements in sample introduction methods(HPLC, surface desorption, etc.), improved capacity for ionization ofeven the largest macromolecules (ESI, MALDI/SELDI) and rapidcomputerized handling of large data sets and comparison topeptide/protein reference libraries, have made mass spectrometry ageneral and powerful tool for automated, large-scale, high-throughputstatic proteomics.

Identification of a Plurality of Proteins or Organic Molecules

The plurality of proteins or organic molecules is analyzed by massspectrometry, using standard methods well known in the art. Thefollowing references describe the application of static massspectrometric techniques to protein identification, with respect toproteome analysis in particular: Ideker T, Thorsson V, Ranish J A,Christmas R, Buhler J, Eng J K, Bumgamer R, Goodlett D R, Aebersold R,Hood L “Integrated genomic and proteomic analyses of a systemicallyperturbed metabolic network.” Science. 2001 May 4; 292 (5518): 929-34;Gygi S P, Aebersold R. “Mass spectrometry and proteomics.” Curr OpinChem. Biol. 2000 October; 4 (5): 489-94; Gygi SP, Rist B, Aebersold R“Measuring gene expression by quantitative proteome analysis” Curr OpinBiotechnol. 2000 August; 11 (4): 396-401; Goodlett D R, Bruce J E,Anderson G A, Rist B, Pasatolic L, Fiehn O, Smith R D, Aebersold R.“Protein identification with a single accurate mass of a cysteinecontaining peptide and constrained database searching” Anal Chem. 2000Mar. 15; 72 (6): 1112-8; and Goodlett D R, Aebersold R, Watts J D“Quantitative in vitro kinase reaction as a guide for phophoproteinanalysis by mass spectrometry” Rapid Commun Mass Spectrom. 2000; 14 (5):344-8; Zhou, H. et al (April 2001) Nature Biotechnol. 19: 375-378.

Protein biochips, also known as protein arrays or antibody arrays, areused to identify proteins. These biochips hold the potential to measureprotein-protein interactions, protein-small molecule interactions, andenzyme-substrate reactions. They can also distinguish the proteins of ahealthy cell from those of a diseased cell. Protein biochips draw on theDNA chip technology developed for genomics and are also able to analyzethousands of samples simultaneously. While the human genome may contain100,000 genes, post-translational modifications and RNA splicing eventsresult in far greater than 100,000 proteins.

Some biochips incorporate a type of mass spectrometry called surfaceenhanced laser desorption/ionization [SELDI], and biochip technology ina single, integrated platform, allowing the proteins to be captured,separated, and quantitatively analyzed directly on the chip. The chipsare read directly by the SELDI process without radioactive orfluorescent labels or genetically engineered tags.

While these techniques are useful, they merely provide a staticassessment of the proteome, not a dynamic assessment of the proteome.The methods herein provide such a dynamic assessment.

Identification of a Plurality of Organic Metabolites

The sample containing organic metabolites is analyzed by massspectrometry, using standard methods well known in the art. Thefollowing reference describes the application of static massspectrometric techniques to metabolite identification, with respect toorganic metabolite identification: Wolfe, R. R. Radioactive and StableIsotope Tracers in Biomedicine: Principles and Practice of KineticAnalysis. John Wiley & Sons; (March 1992).

The pattern of intermediary metabolites and their concentrations inliving systems represents a still-higher level of biochemical phenotype.Myriad organic molecules are present in living systems and serve assubstrates for the enzymes that control flows through functionalbiochemical pathways. A plurality of organic metabolites may be mosteffectively accomplished by use of ESI-MS/MS or GC/MS approaches.

Spots of blood or urine are introduced into an MS device andcharacterized by chromatographic behavior and mass spectrum.

This approach has been used for diagnostic screening of urine samplesfor a wide range of organic metabolites to detect inborn errors ofmetabolism in children. Plant biochemists have also reported somemetabolic profiling work concerning the functional biochemistry ofplants.

Technically, it is easier to isolate organic metabolites than proteinsusing traditional mass spectrometers (e.g. GC/MS instruments) becauseorganic metabolites are generally of smaller size and greater volatilitythan proteins. Other features of organeomics strongly support thepotential applicability of mass spectrometry (e.g. a very large numberof analytes that need to be measured concurrently; requirement tomeasure concentrations; the need for automation and complex datahandling; and the need for comparison to informatics libraries).

Measuring Relative and Absolute Mass Isotopomer Abundances

Measured mass spectral peak heights, or alternatively, the areas underthe peaks, may be expressed as ratios toward the parent (zero massisotope) isotopomer. It is appreciated that any calculation means whichprovide relative and absolute values for the abundances of isotopomersin a sample may be used in describing such data, for the purposes of theinvention.

Calculating Labeled: Unlabeled Proportion of Proteins and OrganicMetabolites

The proportion of labeled and unlabeled proteins or organic metabolitesis then calculated. The practitioner first determines measured excessmolar ratios for isolated isotopomer species of a molecule. Thepractitioner then compares measured internal pattern of excess ratios tothe theoretical patterns. Such theoretical patterns can be calculatedusing the binomial or multinomial distribution relationships asdescribed in U.S. Pat. Nos. 5,338,686; 5,910,403; and 6,010,846 whichare hereby incorporated by reference in their entirety. The calculationsmay include Mass Isotopomer Distribution Analysis (MIDA). Variations ofMass Isotopomer Distribution Analysis (MIDA) combinatorial algorithm arediscussed in a number of different sources known to one skilled in theart. The method is further discussed by Hellerstein and Neese (1999), aswell as Chinkes, et al. (1996), and Kelleher and Masterson (1992), andU.S. patent application Ser. No. 10/279,399, all of which are herebyincorporated by reference in their entirety.

In addition to the above-cited references, calculation softwareimplementing the method is publicly available from Professor MarcHellerstein, University of California, Berkeley.

The comparison of excess molar ratios to the theoretical patterns can becarried out using a table generated for a protein of interest, orgraphically, using determined relationships. From these comparisons, avalue, such as the value p, is determined, which describes theprobability of mass isotopic enrichment of a subunit in a precursorsubunit pool. This enrichment is then used to determine a value, such asthe value A_(X)*, which describes the enrichment of newly synthesizedproteins for each mass isotopomer, to reveal the isotopomer excess ratiowhich would be expected to be present, if all isotopomers were newlysynthesized.

Fractional abundances are then calculated. Fractional abundances ofindividual isotopes (for elements) or mass isotopomers (for molecules)are the fraction of the total abundance represented by that particularisotope or mass isotopomer. This is distinguished from relativeabundance, wherein the most abundant species is given the value 100 andall other species are normalized relative to 100 and expressed aspercent relative abundance. For a mass isotopomer M_(X),

${{{Fractional}\mspace{14mu}{abundance}\mspace{14mu}{of}\mspace{14mu} M_{x}} = {A_{x} = \frac{{Abundance}\mspace{14mu} M_{x}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}}}},$where 0 to n is the range of nominal masses relative to the lowest mass(M₀) mass isotopomer in which abundances occur.

${{\Delta\mspace{14mu}{Fractional}\mspace{14mu}{abundance}\mspace{14mu}\left( {{enrichment}\mspace{14mu}{or}\mspace{14mu}{depletion}} \right)} = {{\left( A_{x} \right)_{e} - \left( A_{x} \right)_{b}} = {\left( \frac{{Abundance}\mspace{14mu} M_{x}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}} \right)_{e} - \left( \frac{{Abundance}\mspace{14mu} M_{x}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}} \right)_{b}}}},$

where subscript e refers to enriched and b refers to baseline or naturalabundance.

In order to determine the fraction of polymers that were actually newlysynthesized during a period of precursor administration, the measuredexcess molar ratio (EM_(X)) is compared to the calculated enrichmentvalue, A_(X)*, which describes the enrichment of newly synthesizedbiopolymers for each mass isotopomer, to reveal the isotopomer excessratio which would be expected to be present, if all isotopomers werenewly synthesized.

Calculating Molecular Flux Rates

The method of determining rate of synthesis includes calculating theproportion of mass isotopically labeled subunit present in the proteinprecursor pool, and using this proportion to calculate an expectedfrequency of a protein containing at least one mass isotopically labeledsubunit. This expected frequency is then compared to the actual,experimentally determined protein isotopomer frequency. From thesevalues, the proportion of protein which is synthesized from addedisotopically labeled precursors during a selected incorporation periodcan be determined. Thus, the rate of synthesis during such a time periodis also determined.

A precursor-product relationship is then applied. For the continuouslabeling method, the isotopic enrichment is compared to asymptotic(i.e., maximal possible) enrichment and kinetic parameters (e.g.,synthesis rates) are calculated from precursor-product equations. Thefractional synthesis rate (k_(s)) may be determined by applying thecontinuous labeling, precursor-product formula:k _(s)=[In(1−f)]/t,

where f=fractional synthesis=product enrichment/asymptoticprecursor/enrichment

and t=time of label administration of contacting in the system studied.

For the discontinuous labeling method, the rate of decline in isotopeenrichment is calculated and the kinetic parameters of proteins arecalculated from exponential decay equations. In practicing the method,biopolymers are enriched in mass isotopomers, preferably containingmultiple mass isotopically labeled precursors. These higher massisotopomers of the proteins, e.g., proteins containing 3 or 4 massisotopically labeled precursors, are formed in negligible amounts in theabsence of exogenous precursor, due to the relatively low abundance ofnatural mass isotopically labeled precursor, but are formed insignificant amounts during the period of protein precursorincorporation. The proteins taken from the cell, tissue, or organism atthe sequential time points are analyzed by mass spectrometry, todetermine the relative frequencies of a high mass protein isotopomer.Since the high mass isotopomer is synthesized almost exclusively beforethe first time point, its decay between the two time points provides adirect measure of the rate of decay of the protein.

Preferably, the first time point is at least 2-3 hours afteradministration of precursor has ceased, depending on mode ofadministration, to ensure that the proportion of mass isotopicallylabeled subunit has decayed substantially from its highest levelfollowing precursor administration. In one embodiment, the followingtime points are typically 1-4 hours after the first time point, but thistiming will depend upon the replacement rate of the biopolymer pool.

The rate of decay of the protein is determined from the decay curve forthe three-isotope protein. In the present case, where the decay curve isdefined by several time points, the decay kinetics can be determined byfitting the curve to an exponential decay curve, and from this,determining a decay constant.

Breakdown rate constants (k_(d)) may be calculated based on anexponential or other kinetic decay curve:k _(d)=[−In f]/t.

While the invention has been described with respect to specific massisotopes and proteins, it will be appreciated how the method can be usedto determine subunit pool composition, and rates of synthesis and decayfor substantially any biopolymer which is formed from two or moreidentical subunits which can be mass isotopically labeled. Similarconsiderations apply for organic metabolites.

Uses of the Techniques of the Present Invention

Examples of medically relevant metabolic determinations which can bemade, using the methods of the invention include: i) molecular fluxrates of proteins involved in fat or cholesterol synthesis in a cell,tissue, or organism, to determine nutritional effects and/or the effectsof drug treatment; ii) molecular flux rates of plasma proteins, as mayoccur in certain disease states before, during and after treatment withvarious drugs; iii) muscle protein dynamics, to determine effects ofsuch determinants as exercise, hormones, drug treatment, age and diseaseon synthesis and breakdown of muscle protein; iv) rates of proteinsynthesis, including viral replication rates in vivo, for assessment ofantiviral drugs on such rates in vivo, and rates of protein synthesisand degradation in a tumor, to determine the efficacy of chemotherapy;v) study of changes in gluconeogenesis, as may be affected by diseasessuch as diabetes, cancer and hypoglycemia. Normal tissue and diseasedtissue can often be distinguished by the types of active genes and theirexpression levels; furthermore, the progression of disease can bedetermined by knowing the rate of change of protein synthesis orbreakdown. Such testing can be performed in vivo directly on humansubjects or ex vivo using cell cultures. Cell cultures may includeanimal cells such as human cells, plant cells, microbial cells includingfungi, yeast and bacteria.

Altered expression patterns of oncogenes and tumor suppressor genes, forexample, are reflected in changes in the synthesis and/or breakdownrates of the proteins that they code, and can effect dramatic changes inthe expression profiles of numerous other genes. Different proteinturnover or organic metabolite fluxes can serve as markers of thetransformed state and are, therefore, of potential value in thediagnosis and classification of tumors. Differences in gene expression,which are not the cause but rather the effect of transformation, may beused as markers for the tumor stage. Thus, the assessment of the proteinkinetic consequences of known tumor-associated genes has the potentialto provide meaningful information with respect to tumor type and stage,treatment methods, and prognosis. Furthermore, new tumor-associatedgenes may be identified by systemically correlating their functionalconsequences on protein or organic metabolite fluxes with their level ofgene expression in tumor specimens and control tissue. Genes whoseexpression is increased or reduced in tumors relative to normal cells,in association with altered fluxes of proteins or organic metabolites,are candidates for classification as oncogenes, tumor suppressor genesor genes encoding apoptosis-inducing products. Generally, the underlyingpremise is that the profiles of dynamic protein or organic metabolitefluxes may provide more specific, direct and accurate markers of genefunction than cruder, less biochemical and less systematic markers ofphenotype can provide. By this means, the physiological function ormalfunction of the gene product in the organism can be established—i.e.true “functional genomics” become possible.

These practical applications can help physicians reduce health carecosts, achieve rapid therapeutic benefits, limit administration ofineffective yet toxic drugs, and monitor changes in (e.g., decreases in)pathogenic resistance.

Kits

The invention provides kits for measuring and comparing molecular fluxrates in vivo. The kits may include isotope-labeled precursor molecules,and in preferred embodiments, chemical compounds known in the art forseparating, purifying, or isolating proteins, and/or chemicals necessaryto obtain a tissue sample, automated calculation software forcombinatorial analysis, and instructions for use of the kit.

Other kit components, such as tools for administration of water (e.g.,measuring cup, needles, syringes, pipettes, IV tubing), may optionallybe provided in the kit. Similarly, instruments for Obtaining samplesfrom the cell, tissue, or organism (e.g., specimen cups, needles,syringes, and tissue sampling devices) may also be optionally provided.

ADVANTAGES OF THE PRESENT INVENTION

The field of the current invention relates to the measurement of DynamicProteomics and Dynamic Organeomics—the kinetics (i.e., the molecularflux rates—synthesis and breakdown rates; production and removal) of theexpressed proteins and organic metabolites, respectively, in a livingsystem. The capacity to measure static levels of very large numbers ofproteins and organic metabolites at one time, by use of massspectrometric or 2-dimensional gel electrophoresis profiling techniques,has greatly advanced the field of Static Proteomics and Organeomics.Missing from all current proteomic and organeomic measurements is a keyelement, however: kinetics or dynamic fluxes (i.e. rates of input andoutflow of molecules, which brings in the dimension of time).

1) Differences from current mass spectrometric proteome profilinginclude:

-   -   a) Current static mass spectrometric profiling techniques do not        measure fluxes (kinetics or flow of molecules through pathways).    -   b) The operational procedure of a prior step wherein stable        isotope labels are administered in vivo or ex vivo, before        collection of the biological sample, is not used or known in the        field of profiling for proteomics and organeomics.    -   c) The analytic procedure of monitoring particular mass        isotopomers, measuring their quantitative abundances, and        calculating individualized synthesis and turnover rates for each        molecule based on its molecular formula, the stable isotope        label added and mass isotopomer combinatorial calculations, has        not been used in the field of proteomics and organeomics.    -   d) By adding kinetics, the focus is changed fundamentally from        concentrations of individual molecules to the control of pathway        fluxes into and out of pools of molecules (i.e. to the true        biochemical consequences of individual molecules on functional        biochemical outputs).    -   e) Kinetic measurements allow direct inference of regulatory        steps controlling homeostasis of the proteome and organeome.

2) Fundamental advantages and/or surprising results that have emerged ormay be expected:

-   -   a) The translational (protein synthesis) program of a cell or        organism can be immediately observed, without a lag phase for        change in protein concentrations.    -   b) The protein catabolic program of a cell or organism can be        observed directly, which data is not otherwise available.    -   c) The remarkable result has emerged that labeling of proteome        has up to two orders of magnitude greater sensitivity than        static measurements for detecting treatment effects (i.e. <200        proteins out of 20,000 show large changes in static        concentrations at steady state after even the most potent        interventions, whereas up to 40-50% of proteins show large        changes in synthetic or catabolic rates at steady state after        potent interventions).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit andscope of the appended claims.

Applicants have not abandoned or dedicated to the public any unclaimedsubject matter.

What is claimed is:
 1. A method of determining the molecular flux rates of a plurality of proteins in a cell, tissue or organism, said method comprising: a) administering one or more isotope-labeled protein precursors to said cell, tissue or organism for a period of time sufficient for one or more isotope labels of the one or more isotope-labeled protein precursors to be incorporated into proteins in the cell, tissue or organism; b) obtaining a sample from the cell, tissue, or organism wherein the sample comprises a plurality of proteins; c) degrading the plurality of proteins to form a mixture of peptides from the plurality of proteins; d) performing a first mass spectrometry on the mixture of peptides from the plurality of proteins to identify a plurality of mass isotopomeric envelopes of an initial series of ionic fragments representing individual proteins; e) performing a second mass spectrometry to identify secondary fragments of the initial series of ionic fragments; f) comparing the initial series of ionic fragments identified by the first mass spectrometry with the secondary fragments identified by the second mass spectrometry to identify one or more of the individual proteins in the sample; g) quantifying relative and absolute mass isotopomer abundances of the ionic fragments of one or more of the identified individual proteins within the mass isotopomeric envelope; and h) calculating the molecular flux rates of one or more of the identified individual proteins to determine the molecular flux rates of said plurality of proteins.
 2. The method of claim 1, wherein said administering step (a) is continuous.
 3. The method of claim 1, wherein said administering step (a) comprises administering said one or more protein precursors at regular measured intervals.
 4. The method of claim 1, wherein said one or more isotope-labeled protein precursors are administered orally.
 5. The method of claim 1, further comprising the step of displaying the molecular flux rates of one or more of the identified individual proteins of said plurality of proteins.
 6. The method of claim 1, wherein said one or more isotope-labeled protein precursors is an isotope-labeled amino acid.
 7. The method of claim 6, wherein the one or more isotope-labeled protein precursors are selected from the group consisting of isotope-labeled H₂O, isotope-labeled CO₂, isotope-labeled NH₃, and isotope-labeled HCO₃.
 8. The method of claim 1, wherein said one or more isotope labels is selected from the group consisting of ²H, ¹³C, ¹⁵N,¹⁸O, ³³S and ³⁴S.
 9. The method of claim 8, wherein said one or more isotope label is ²H.
 10. The method of claim 1, wherein said one or more isotope-labeled protein precursor is selected from the group consisting of ²H₂O, H₂ ¹⁸O, ¹³CO₂, C¹⁸O¹⁷O, H¹⁶CO₃, ¹⁵NH₃, ²H-labeled amino acids, ¹³C-labeled amino acids, ¹⁵N-labeled amino acids, ¹⁸O-labeled amino acids, ³⁴S-labeled amino acids, and ³³S-labeled amino acids.
 11. The method of claim 10, wherein said one or more isotope-labeled protein precursor is ²H₂O.
 12. The method of claim 1, wherein the organism is a human.
 13. The method of claim 1, further comprising administering a diagnostic or therapeutic agent to said cell, tissue, or organism prior to said administering step (a).
 14. A method of determining the effects of one or more genes on the molecular flux rates of a plurality of proteins in a cell, tissue, or organism, comprising: a) determining the molecular flux rates a plurality of proteins in a first population of one or more cells, tissues, or organisms according to the method of claim 1, wherein said cells, tissues, or organisms of said first population comprise said one or more genes; b) determining the molecular flux rates of the plurality of proteins in a second population of one or more cells, tissues, or organisms according to the method of claim 1, wherein said second population does not comprise said one or more genes; and c) comparing the molecular flux rates in said first and second populations to determine the effect of one or more genes on the molecular flux rates of a plurality of proteins.
 15. The method of claim 14, further comprising isolating a plurality of samples from said cell, tissue or organism.
 16. The method of claim 1, further comprising isolating a plurality of samples from said cell, tissue or organism.
 17. The method of claim 1, wherein the one or more isotope-labeled protein precursors is one or more stable isotope-labeled protein precursors.
 18. The method of claim 1, wherein the one or more isotope-labeled protein precursors is selected from the group consisting of ²H-labeled amino acids, ¹³C-labeled amino acids, ¹⁵N-labeled amino acids, ¹⁸O-labeled amino acids, ³⁴S-labeled amino acids, and ³³S-labeled amino acids. 